Tumor necrosis factor-alpha (TNF) is an important cytokine regulator of the immune system. Its dysregulation is implicated in causing cancer, inflammatory bowel disease, and other systemic inflammatory disorders, including rheumatoid arthritis. TNF activity leads to the activation of pro inflammatory pathways regulated by the transcription factor NF-?B. TNF inhibitors and monoclonal antibodies are used to treat many of these autoimmune and inflammatory diseases, though with limited efficacy. Significant need exists toward improving anti-TNF based therapies. Enteric bacterial pathogens have evolved mechanisms to subvert efficiently the pro-inflammatory host defenses. Motivated by the need to discover anti-inflammatory compounds that could be developed to treat autoimmune disorders, cell-free supernatants were screened from various enteric pathogens for their ability to inhibit TNF activation of the NF-?B pathway. It was discovered that enterotoxigenic Escherichia coli (ETEC) secretes a heat-stable protein that efficiently blocks the host NF-?B signaling pathway induced by TNF. This proposal will use a combination of biochemical and genetic approaches to identify this TNF antagonist and to subsequently characterize its mechanism of action. In Specific Aim1, a combination of transposon mutagenesis, biochemical fractionation, and mass spectrometry techniques will be used to identify the TNF antagonist and the genetic locus on which it is encoded. In Specific Aim 2, the interaction of the TNF antagonist with the host ubiquitination machinery and with NF-?B regulatory proteins will be characterized. Identifying and characterizing this ETEC protein may advance the future development of novel anti- inflammatory compounds.